This proposal will investigate the biosynthesis of the kutzneride family of natural products, and more specifically the nonproteinogenic amino acid piperazate. Piperazate, the aza-analogue of pipecolate, can serve as a six-membered proline turn mimic and is present in numerous nonribosomal peptide antibiotics in various forms. The availability of tailored proline mimics through biosynthesis may lead to the production of novel peptide natural product analogues. The specific aims focus on the biosynthesis, functionalization, and incorporation of piperazates into the kutzneride scaffold. A better understanding of these processes should lead to the development of excised piperazate elongation domains. Furthermore, usage of these elongation domains may furnish analogues with improved antibiotic or antitumor properties. Research will focus on the enzymatic N-N bond formation and oxidative tailoring of the piperazate entity and the specificity of the proposed piperazate A domain. Determination of the A domain specificity requires synthesis of various precursors and analysis of their incorporation through an ATP-PPi exchange assay with the expressed protein. Subsequent analysis of amino acid incorporation will determine the most likely piperazate derivative activated by the corresponding A domain. These results would aid in the investigation of the timing of piperazate functionalization. Cloning, overexpression, and purification of the proposed PipCAT domain and combination with the known PheATE or 2-hydroxy-3,3-dimethyl butyric acid AKRT domains would yield diketopiperazine and diketomorpholine products whose structure would closely mimic cyclo(Phe-Pro) and cyclo(Leu-Pro), a class of broad-spectrum antimicrobial agents. The conformational similarities of piperazate to that of proline and pipecolate make it a desirable pharmacophore for future research. Currently, very little is known about the biosynthesis and incorporation of piperazates in natural products. Understanding the mechanisms behind the biosynthesis, tailoring, and incorporation of piperazate would allow for the production of various nonribosomal peptide natural products. Ultimately, these findings would produce new methods for re-engineering proteins to furnish new antimicrobial agents. PUBLIC HEALTH RELEVANCE: Bacterial resistance to antibiotics is a longstanding problem in the healthcare field. This proposal focuses on understanding how specific enzymes operate and developing new methods for the production of unique antibiotics through genetic engineering. These advances could potentially lead to new drugs to overcome bacterial resistance.